In the fields of medical, dietary, environmental and chemical sciences there is an increasing need for the selective separation of specific substances in complex mixtures of related substances. The end goal can be the preparative isolation of a certain compound or compounds or measurements of their concentration. Molecularly imprinted polymers (MIPs) often exhibit a high selectivity towards their substrate in analogy with antibody-antigen complementarity. (1) The technique shows promise in chiral separations of, for example, amino acid derivatives, peptides, phosphonates, aminoalcohols and beta-blocking compounds, affinity chromatography of nucleotides and the DNA-bases, as well as substitutes for antibodies in immunoassays for commercial drugs. (2) Molecular imprinting (MI) consists of the following key steps (FIG. 1): (1) Functional monomers are allowed to interact reversibly with a template molecule in solution. (2) The hereby formed template assemblies are copolymerized with a crosslinking monomer resulting in a crosslinked network polymer. (3) The template is displaced and the materials can be used for selective molecular recognition of the corresponding compound. If these are crushed and sieved they can be packed in a chromatographic column and used for chromatographic separation of the template from structurally related analogues. Analytical as well as preparative applications are possible. Preparative applications can be separation of a compound from a complex mixture of structurally related compounds and isolation of the compound. This can be through an affinity chromatographic procedure where pH, ion strength or solvent gradients can be used in order to control the strength of interaction with the stationary phase. The separation can target enantiomers or diastereomers in a mixture of enantiomers or diastereomers of one or many compounds. Analytical applications can in addition to the above mentioned separations be: competitive binding assays, chemical sensors or selective sample enrichments. (3)
Currently the most widely applied technique to generate molecularly imprinted binding sites is represented by the noncovalent route. (4) This makes use of noncovalent self-assembly of the template with functional monomers prior to polymerisation, free radical polymerisation with a crosslinking monomer and then template extraction followed by rebinding by noncovalent interactions. Although the preparation of a MIP by this method is technically simple it relies on the success of stabilisation of the relatively weak interactions between the template and the functional monomers. Stable monomer-template assemblies will in turn lead to a larger concentration of high affinity binding sites in the resulting polymer. The materials can be synthesized in any standard equipped laboratory in a relatively short time and some of the MIPs exhibit binding affinities and selectivities in the order of those exhibited by antibodies towards their antigens. Most MIPs are synthesized by free radical polymerisation of functional monounsaturated (vinylic, acrylic, methacrylic) monomers and an excess of crosslinking di- or tri-unsaturated (vinylic, acrylic, methacrylic) monomers resulting in porous organic network materials. These polymerisations have the advantage of being relatively robust allowing polymers to be prepared in high yield using different solvents (aqueous or organic) and at different temperatures. This is necessary in view of the varying solubilities of the template molecules.
The most successful noncovalent imprinting systems are based on commodity acrylic or methacrylic monomers, such as methacrylic acid (MAA), crosslinked with ethyleneglycol dimethacrylate (EDMA). Initially, derivatives of amino acid enantiomers were used as templates for the preparation of imprinted stationary phases for chiral separations (MICSPs) but this system has proven generally applicable to the imprinting of templates allowing hydrogen bonding or electrostatic interactions to develop with MAA. (5) The procedure applied to the imprinting with L-phenylalanine anilide (L-PA) is outlined in FIG. 1. In the first step, the template (L-PA), the functional monomer (MAA) and the crosslinking monomer (EDMA) are dissolved in a poorly hydrogen bonding solvent (diluent) of low to medium polarity. The free radical polymerisation is then initiated with an azo initiator, commonly azo-N,N′-bis-isobutyronitrile (AIBN) either by photochemical homolysis below room temperature or thermochemically at 60° C. or higher. In the final step, the resultant polymer is crushed by mortar and pestle or in a ball mill, extracted in a Soxhlet apparatus and sieved to a particle size suitable for chromatographic (25-38 μm) or batch (150-250 μm) applications. The polymers are then evaluated as stationary phases in chromatography by comparing the retention time or capacity factor (k′) of the template with that of structurally related analogues.
A number of compound classes are only poorly recognized by polymers prepared using the present imprinting protocols. Furthermore the binding strength between the functional monomer and the template is often insufficient, leading to a low sample load capacity and significant non-specific binding. There is therefore a need for the development of new functional monomers binding more strongly to the template and allowing recognition of new compound classes. For instance monomers designed to bind carboxylic-, phosphoric- or phosphonic-acid templates are needed. A few examples are given here. One goal in the analytical applications is the development of imprinted polymers capable of strongly binding nucleotides and discriminating between the four bases. This may lead to new methods for sensitive detection of modified DNA bases and may thus find use in early cancer diagnosis. However, this requires development of new functional monomers capable of binding phosphate thereby increasing the organic solubility of the template. Moreover recognition of adducts of the four DNA bases formed upon exposure of DNA to oxidizing or alkylating agents is one important goal in the development of methods aiming at early diagnosis or prediction of cancer risks. However, this requires development of new functional monomers capable of binding purine and pyrimidine bases with high fidelity. In chiral separations and for analytical applications using imprinted polymers there is also a need for functional monomers capable of strongly binding compounds with weakly acidic hydrogens such as in alcohols, imides, sulfonamides, phosphonamides, ureas including important classes like carbohydrates, sulfonylureas, hydantoins, barbiturates, purine, pyrimidine and pteridine bases. If these were available, higher selectivities and capacities for the target compound or enantiomer could be expected. Weakly polar or nonpolar compounds are also an important group of targets that are difficult to bind with high selectivity. Among these are highly important and relevant analytes such as dioxines (tetrachlorodioxodibenzene, TCDB), polyaromatic hydrocarbons (PAHs), aldehydes, halogenated hydrocarbons and phosphonates (such as those found in nerve agents and insecticides).
This invention describes the synthesis and use of new classes of functional monomers for molecular imprinting. Two classes are based on the amidine-functional group and can be synthesized with various basicities, hydrophobicities and chiralities. Amidine functionalized monomers have been previously used in molecular imprinting in the form of derivatives of 4-vinylbenzamidine. (16) These are synthesized in several steps and can only be produced with a limited structural variation. Also no chiral amidine monomers have been reported for use in molecular imprinting. This invention introduces a new class of amidine based monomers, formamidines, that are accessible in high yield in only a few synthetic steps. The monomers are suited for imprinting of a variety of functional groups including carboxylic and phosphorous acids, alcohols, imides, sulfonamides, phosphonamides and ureas which can be achiral or chiral. Furthermore the synthetic route allows their properties such as pKa values and polarities to be easily tuned. The other class of amidine based monomers are chiral amidines that potentially will enhance the recognition of chiral substrates. This basic benzamidine unit has previously been used as chiral shift reagents in determination of optical purity by NMR. (17) Another class of functional monomers are vinyl-methacrylolyl- or acryoloyl-based alkyl or aryl diamines (4) which was previously used as a chiral shift reagent for diols. This monomer is designed to bind carbohydrates.
Other classes of polymerisable functional monomers of the present invention are receptor analogue monomers, urea based monomers, thiourea based monomers, purine or pteridine based monomers, acrylamido or methacrylamido pyridine monomers, aminovinylpyridine monomers, strong hydrogen bond accepting monomers that are unable to function as hydrogen bond donors, such as hexamethyl phosponamide based monomers, and strong hydrogen bond donating monomers that are unable to function as hydrogen bond acceptors, such as N,N′-disubstituted phenyl urea monomers, as defined below.
This invention describes the synthesis and use of new classes of functional monomers for molecular imprinting. They will be described below together with a number of non-restricting examples of their synthesis and use.
The invention will now be described in more detail giving a number of non-restricting examples: